INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Inhalation of crystalline silica dusts leads to silicosis, a chronic lung pathological process characterized by granulomatous inflammation and fibronodular response (1). The course of the human disease is often insidious, and progression occurs in the absence of continued exposure to silica, leading to chronic respiratory insufficiency and even death.

The disease has been reproduced in several animal models, which develop morphologic changes similar to the human disorder (2, 3). Silica-induced granulomatous inflammation is characterized by accumulations of macrophages and lymphocytes with mineral particles in the nodules. The inflammatory reaction is followed by a fibrotic response with fibroblast proliferation and excessive extracellular matrix accumulation, including interstitial collagens and insoluble elastin (2, 4, 5). However, the mechanisms responsible of the pathological alterations leading to the fibrotic response in the silicotic nodules are far from being elucidated.

Normal extracellular matrix remodeling depends on a balanced synthesis/degradation pattern. Previous work has found evidence of increased collagen expression and synthesis (2, 4) but research on the breakdown pathway is scanty.

Extracellular matrix degradation is a complex multistep process that involves a family of zinc-dependent endopeptidases known as matrix metalloproteinases (MMPs). MMPs consist of a number of structurally related enzymes capable of digesting extracellular matrix and basement membrane components (6, 7). At least 18 family members having common propeptide and catalytic domains have been identified. By considering the domain structure and/or substrate affinity, four major subgroups have been classified including collagenases, stromelysins, gelatinases, and membrane-type metalloproteinases. Collagenases (MMP-1, MMP-8, and MMP-13) degrade mainly fibrillar collagens. Stromelysins (MMP-3, MMP-10, MMP-11, and MMP-20) digest proteoglycans and some glycoproteins. Gelatinases (MMP-2 and MMP-9) have a substrate affinity for basement membrane type IV collagen, denatured collagens (gelatin), and elastin. MMP-7 and MMP-12 share substrate affinity with gelatinases although they have a different domain structure. Finally, the membrane-type metalloproteinases (MT1-, MT2-, MT3-, and MT4-MMPs) have a broad substrate affinity including progelatinase A activation (8). MMP activity is regulated at different levels, including transcriptional control, extracellular activation of proenzymes, and active enzyme inhibition. The tissue inhibitors of metalloproteinases (TIMPs) are a family with, currently, four members identified; they form complexes with active enzymes and inhibit MMP activity (8, 9).

Because during fibrosis there is a progressive deposit of collagens, we hypothesized that a different balance of MMPs/TIMPs should occur in the inflammatory phase in comparison with the fibrotic phase of the development of silicotic granulomas.

Therefore, the present study was designed to compare the expression of interstitial collagenase 3 (MMP-13), gelatinases A and B (MMP-2 and MMP-9), as well as TIMP-1 and TIMP-2 in early (15 d) and late (60 d) silicotic granulomas.

	METHODS

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Experimental Model

Lung silicosis was induced in pathogen-free male Wistar rats (200-250 g) by a single intratracheal administration of 50 mg of quartz dust in sterile saline (dispersity less than 5 µm). Food and water were available ad libitum throughout the experimental period. Eight rats were sacrificed at 15 and 60 d after silica instillation, and eight normal animals instilled intratracheally with saline were used as controls. Animals were anesthetized and the right lung was instilled with 4% paraformaldehyde and used for histology, in situ hybridization, and immunohistochemistry. The left lung was used for lung tissue zymography.

The extent of the lung granulomatous lesions was analyzed by a semiquantitative assessment as described previously (10). Briefly, two hematoxylin and eosin (H&E)-stained slides per animal were scanned completely in a zigzag fashion at ×32 magnification, and the extent of the lesion was determined as the percentage of the lung occupied by well-formed nodules.

Bronchoalveolar Lavage

In a parallel experiment, bronchoalveolar lavage was performed in six controls and six silica-exposed rats at 15 and 60 d. Lungs were lavaged by flushing with three 8-ml aliquots of sterile saline solution at 37° C through a tracheal cannula. Seventy to 80% of the instilled volume was recovered without significant differences between silicotic and control rats. Total proteins were measured by the Bradford method (11). Total cell counts were determined in samples of unfractionated bronchoalveolar lavage fluid (BALF), using a hemocytometer counting chamber. The BALF was then centrifuged at 400 g for 10 min at 4° C. Cells were fixed in 50% ethyl alcohol and 2% Carbowax (50% polyethylene glycol) and centrifuged at 400 g for 15 min, and several slides per sample were stained with hematoxylin and eosin and used for differential cell counting.

Lung Tissue and BALF Gelatin Zymography

Lung samples (20 mg/ml) from control rats and from rat 15 and 60 d after silica instillation were homogenized in 10 mM 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS)-20 mM HEPES (pH 7.5)-150 mM NaCl. After centrifugation, supernatant aliquots containing 5 µg of protein were used to analyze lung tissue gelatinase activity. For BALF zymograms equal volumes of lavage fluid (8-12 µl) containing ~ 0.5 to 0.7 µg of protein (control rats) and ~ 2.8 to 3.4 µg of protein (rats 15 and 60 d after silica exposure) were mixed with an equal volume of Laemmli sample buffer containing 3% sodium dodecyl sulfate (SDS) (12). Gelatinase activity was detected in a gelatin substrate SDS gel as previously described (13). Serum-free conditioned medium from human lung fibroblasts was used as a gelatinase A marker, and serum-free conditioned medium from phorbol myristate acetate (PMA)-stimulated U2-OS cells was used as a marker of gelatinase B. Similar gels were incubated but in the presence of 20 mM EDTA. The molecular weight of the gelatinolytic bands was estimated by comparison with prestained molecular weight markers (Bio-Rad, Hercules, CA).

Determination of Collagen Content

Lung fragments from control rats and from rats exposed to silica for 15 and 60 d were hydrolyzed in 6 N HCl for 24 h at 110° C, and hydroxyproline colorimetric analysis was performed as described by Woessner (14). Collagen content was expressed as micrograms per milligram of lung tissue, after multiplication of hydroxyproline amount by 7.23.

In Situ Hybridization

Riboprobes for in situ hybridization were generated as previously described (13) from human cDNA gelatinase A, provided by G. I. Goldberg (Washington University, St. Louis, MO); rat cDNA collagenase, provided by C. O. Quinn (St. Louis University, St. Louis, MO); rat cDNA gelatinase B for the 3' region, donated by J. Windsor (University of Alabama at Birmingham, Birmingham, AL). Mouse TIMP-1 and TIMP-2 cDNA were kindly donated by D. Edwards (University of Calgary, Alberta, Canada).

In situ hybridization was performed on 4-µm sections as previously reported, using digoxigenin-labeled riboprobes (13, 15). Three different sections from controls and silicotic animals were assayed. Some sections were hybridized with digoxigenin-labeled sense RNA probes. After hybridization, tissues were incubated with a polyclonal sheep anti-digoxigenin antibody coupled to alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN) for 1 h at room temperature. The color reaction was performed by incubation in 100 mM Tris-HCl-50 mM MgCl₂ (pH 9.5) with 0.1 mM levamisole, 4-nitroblue tetrazolium chloride (0.338 mg/ml), and 0.173 mg/ml 5-bromo-4-chloro-3-indolylphosphate or Fast Red chromogen (Biomeda, Foster City, CA). Sections were lightly counterstained with eosin when nitroblue tetrazolium was used, or with hematoxylin for Fast Red-treated slides.

Immunohistochemistry

Rabbit polyclonal anti-rat collagenase and mouse monoclonal antibody against rat gelatinase B were kindly provided by J. Windsor. Anti-human TIMP-2 monoclonal antibody that cross-reacts with rat TIMP-2 was from Fuji Chemical Industries (Tokyo, Japan). Macrophages were detected using monoclonal mouse anti-human macrophage HAM56 antibody (7.5 µg/ml; Dako, Carpinteria, CA). Mesenchymal cells were revealed with monoclonal mouse anti-vimentin antibody (9.5 µg/ml; Dako).

Immunoreactive proteins were identified in tissue sections, as previously described (13, 15). Briefly, slides were deparaffinized in xylene, rehydrated, and heated for 5 min in a microwave oven in 10 mM citrate buffer, pH 6.0. Three different sections from controls and silicotic rats were incubated at 4° C overnight with primary antibodies at the following concentrations: 50 µg/ml of anti-MMP-13, anti-MMP-9, or anti-TIMP-2; 7.5 µg/ml of anti-HAM56; and 9.5 µg/ml of anti-vimentin. A secondary biotinylated antibody (Vector Laboratories, Burlingame, CA) and avidin-biotin peroxidase complex were applied sequentially followed by 3,3'-diaminobenzidine in phosphate-buffered saline (PBS) containing 0.05% H₂O₂. The sections were counterstained with hematoxylin. The primary antibodies were replaced with normal rabbit or mouse sera for negative control slides.

Statistical Analysis

For statistical comparison, a two-tailed Student t test for unpaired observations was used. A p value < 0.05 was considered significant.

	RESULTS

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Bronchoalveolar Lavage Fluid

The mean volume recovery of BALF after the instillation of saline solution or silica was ~ 75%, and there were no differences between normal and silica-exposed rats. Regarding protein content, a threefold increase in total protein was observed in silicotic rats as compared with saline instilled animals (81 ± 25 versus 280 ± 61 µg/ml at 15 d and 85 ± 28 versus 210 ± 40 µg/ml at 60 d, p < 0.01).

The number and types of cells obtained in the lung lavage are shown in Figure 1. At 15 and 60 d after silica instillation a significant increase in total lung cells was observed when compared with control samples (p = 0.001). The type of inflammatory cells in BALF control rats consisted mostly of macrophages (88 ± 3.4%) and lymphocytes (10 ± 4.1%). At 15 d after silica exposure, a significant increase in the absolute number of lymphocytes and neutrophils was noticed. At 60 d after silica instillation, in addition to these inflammatory cells, an increase in BALF macrophages was also observed. Macrophages and neutrophils were significantly higher at 60 d compared with 15 d (3.11 ± 0.63 × 10⁶ versus 1.42 ± 0.28 × 10⁶, p < 0.0001, and 1.93 ± 0.52 × 10⁶ versus 0.79 ± 0.41 × 10⁶, p < 0.005, respectively). Eosinophils were rarely seen.

View larger version (25K): [in this window] [in a new window]   Figure 1.   Cellular profile of bronchoalveolar lavage fluid from control rats and silica-exposed animals after 15 and 60 d. Total cell counts were determined on fresh BALF, and differential cell counts were assessed using hematoxylin and eosin staining. Results are expressed as means ± SD.

Histopathology

Lung histological examination of animals on Days 15 and 60 after silica instillation revealed numerous granulomas containing silica, mostly with a peribronchiolar distribution. Representative lung tissue sections from rats instilled with silica are shown in Figure 2. Fifteen days after exposure, granulomas were prominently cellular and slight to moderate collagen deposition was observed in some of them by using picrosirius red staining (Figure 2A). After 60 d, the fibrotic response progressed and a fibrillar collagenous framework was observed mainly in the center of the granulomas (Figure 2B), whereas inflammatory cells and fibroblasts were prominent in the peripheral zone. However, new and small inflammatory nodules were also noticed at this time. Often, granulomatous lesions at 60 d of evolution were larger as the result of confluence of two or more nodules. The extension of lung parenchyma occupied by silicotic granulomas was significantly higher at 60 d (57.5 ± 11.7 versus 30 ± 5.8% at 15 d, p < 0.001). Alveolar foamy macrophages were usually present in the adjacent zones of granulomatous response either at 15 or 60 d after silica instillation. These findings might explain the increased prominence of BALF inflammatory cells at 60 d.

View larger version (154K): [in this window] [in a new window]   Figure 2.   Light photomicrographs of sections of lung tissue 15 d (A) and 60 d (B) after silica instillation (original magnification ×2.5). Insets: Photomicrographs, taken under polarized light, of granulomas stained with picrosirius red. Collagen can be seen as brightly birefringent fibers.

Lung Collagen Content

When compared with control animals, 15-d silica-exposed rats showed no difference in lung collagen content expressed as micrograms of collagen per milligram of dry weight (88.2 ± 20.8 versus 100.4 ± 36 µg/ml). By contrast, at 60 d after silica instillation lung collagen content was significantly increased (88.2 ± 20.8 versus 170.2 ± 34.4 µg/ml, p = 0.01).

Lung Expression and Localization of Matrix Metalloproteinases at 15 d

Early silicotic granulomas (15 d) revealed an intense staining for all MMPs and TIMPs assayed. Labeling was restricted to the granulomas and surrounding areas while nongranulomatous parenchyma exhibited only scattered positive cells for MMP or TIMP mRNA. Saline-treated animals did not show meaningful signals in the lung parenchyma.

Collagenase 3 transcript analyzed by in situ hybridization was detectable in the peripheral zone of the granulomas, as well as in the surrounding parenchyma (Figure 3B). In these areas, an intense positive signal was primarily seen in the cytoplasm of alveolar macrophages and interstitial cells (Figure 3D). Immunohistochemistry confirmed the presence of numerous collagenase-producing macrophages in peribronchiolar areas of early granulomatous lesions (Figure 3E). Inside the granulomas, immunoreactive collagenase 3 was also noticed in macrophages, alveolar epithelial cells of alveoli trapped in the granulomas, and cells coincident with vimentin-positive mesenchymal cells (Figures 3G-3I). Controls using sense riboprobe and nonspecific antisera displayed no reactivity (Figures 3C-3F). Normal lungs did not show a detectable signal (Figure 3A).

View larger version (84K): [in this window] [in a new window]   Figure 3.   Lung expression and localization of collagenase 3 15 d after silica instillation. (A) In situ hybridization (ISH) of digoxigenin- labeled antisense probe in normal lung. (B) ISH in silicotic rat. Note the intense signal in the peripheral zone of the granuloma. (C ) ISH of the same lung field, using the sense riboprobe. (D) High-power magnification showing a cluster of positively stained alveolar macrophages in the surrounding area of a silicotic nodule. (E) Immunoreactive collagenase revealed by diaminobenzidine staining, showing areas of positive alveolar macrophages in a peribronchiolar region. (F ) Negative control section of the same field, showing absence of collagenase 3 immunostaining. (G) Immunohistochemical localization of collagenase 3-positive cells inside a granuloma. (H ) High-power magnification showing putative inflammatory and fibroblast-like positive cells inside a granuloma. (I ) Same granuloma, showing vimentin-positive cells in a silicotic nodule. (A-D) Lightly counterstained with eosin; (E-I ) counterstained with hematoxylin. Original magnification: (A) ×40; (B and C ) ×2.5; (D) ×100; (E and F ) ×10; (G) ×20; (H ) ×100; (I ) × 20.

In situ hybridization of digoxigenin-labeled antisense riboprobe for gelatinase A revealed positive staining often localized in the central area of the granulomas and in the neighboring areas, where an inflammatory response was evident (Figure 4A). In some other granulomas the label was noticed in a diffuse pattern (Figure 4B). MMP-2 mRNA was localized in cells resembling mesenchymal cells, identified in parallel slides by vimentin staining (not shown), and in macrophages (Figure 4C). Normal lung showed scattered positive cells (Figure 4D). No signal was observed when sense riboprobe was used (Figure 4E).

View larger version (126K): [in this window] [in a new window]   Figure 4.   Lung expression and localization of gelatinase A at 15 d. (A and B) ISH of gelatinase A transcript, showing positive cells inside the granulomas and surrounding areas. (C ) High-power magnification exhibiting positive macrophage-like cells for gelatinase A mRNA transcript. (D) ISH for normal lung. (E ) ISH with sense riboprobe.

Gelatinase B mRNA was observed in the peripheral area of some silicotic granulomas as well as in a central localization in some others (Figure 5B). In situ hybridization revealed positive alveolar macrophages, neutrophils, and putative alveolar epithelial cells, some of them located in the corners of alveoli protruding to the alveolar spaces (Figures 5C and 5D). Immunoreactive protein was also localized in alveolar macrophages, and inside the granulomas in presumed macrophages, determined by HAM56 coincident staining of parallel sections (Figures 5F-5I). No signal was observed when tissue samples were hybridized with the sense probes or nonimmune sera (Figures 5E and 5J).

View larger version (85K): [in this window] [in a new window]   Figure 5.   Lung expression and localization of gelatinase B at 15 d. (A) ISH of normal lung. (B) ISH showing positive signal localized inside silicotic granulomas and surrounding area. (C ) mRNA transcript of gelatinase B in alveolar macrophages. (D) Same signal in neutrophils and epithelial cells. (E ) ISH with sense riboprobe. (F  ) Immunoreactive gelatinase B in the interior of a silicotic nodule. (G) Higher magnification showing macrophage-like cells. (H ) Immunoreactive gelatinase B localized in alveolar macrophages. (I ) Same granuloma as in (F  ) and (G) showing cells staining positive for HAM56. ( J ) Negative control section showing absence of gelatinase B immunostaining. (A, B, and E ) Lightly counterstained with eosin; (C, D, F-I ) counterstained with hematoxylin.

Lung Expression and Localization of Tissue Inhibitors of Metalloproteinases at 15 d

Light microscopic in situ hybridization with TIMP-1 antisense riboprobe revealed intense labeling in the granulomatous lesions (Figures 6A-6C). TIMP-1 mRNA signal was observed in cells coincident with vimentin staining, putatively identified as mesenchymal cells, and in areas corresponding to macrophage-like cells as compared with parallel sections stained with HAM56 (not shown). TIMP-2 transcript revealed a distribution similar to that of TIMP-1 mRNA inside the granulomas, as well as in the macrophages of the neighboring areas (Figures 6E and 6F). Immunoreactive TIMP-2 protein was noticed in clusters of fibroblast-like cells inside the granulomas (Figures 6I and 6J) and in cuboidal epithelial cells in alveoli trapped in the nodule (Figure 6K). Normal lungs showed no signal or scattered positive cells as illustrated for TIMP-1 mRNA (Figure 6D) or TIMP-2 immunoreactive protein (Figure 6L). Controls using TIMP-2 and TIMP-1 sense riboprobes showed no reactivity (Figures 6G and 6H).

View larger version (133K): [in this window] [in a new window]   Figure 6.   Lung expression and localization of TIMP-1 and TIMP-2 at 15 d. (A) Tissue section hybridized with digoxigenin-labeled TIMP-1 antisense RNA probe. (B) ISH showing TIMP-1 transcript in numerous cells inside a silicotic granuloma. (C ) High magnification of an area illustrating positive staining in inflammatory and mesenchymal-like cells. (D) Normal lung hybridized with TIMP-1 mRNA. (E ) Silicotic lung hybridized with digoxigenin-labeled TIMP-2 antisense riboprobe. (F  ) ISH showing TIMP-2 transcript in alveolar macrophages located in alveolar walls and spaces in a granuloma surrounding area. (G and H ) ISH with TIMP-2 and TIMP-1 sense riboprobes. (I ) Immunohistochemical localization of TIMP-2 in the interior of a silicotic nodule. ( J ) Immunoreactive TIMP-2 in fibroblast-like cells. (K ) Immunoreactive TIMP-2 in epithelial and inflammatory cells. (L) No immunoreactive protein is noticed in a normal lung. (A, D-H ) Lightly counterstained with eosin; (B, C, I-L) counterstained with hematoxylin.

Expression of Collagenase 3, Gelatinases A and B, and TIMP-1 and TIMP-2 at 60 d

Matrix metalloproteinase expression in 60-d silicotic granulomas was markedly lower than that observed in the early lesions. By in situ hybridization, positive label was detected in a few areas in a nonuniform distribution as noted for collagenase 3, gelatinase A, and gelatinase B (Figures 7A-7C, respectively). When highly fibrotic nodules were evaluated, as revealed by Masson's staining (Figure 7E), scarce signal, or none at all, was found inside the granuloma as exemplified for gelatinase A labeling (Figure 7D). Absence of signal could not be attributed to a noncellular scar because the granuloma showed numerous cells as revealed by hematoxylin and eosin staining of the same granuloma at higher magnification (Figure 7F).

View larger version (136K): [in this window] [in a new window]   Figure 7.   Expression of metalloproteinases and TIMPs 60 d after silica instillation. (A) ISH exhibiting collagenase 3 mRNA transcript. (B) ISH showing gelatinase A mRNA. (C ) ISH revealing gelatinase B transcript. (D) Tissue sections hybridized with digoxigenin-labeled gelatinase A antisense RNA probe, showing scarce signal in silicotic granulomas. (E ) Masson's taining of same granuloma as in (D), revealing highly fibrotic nodule. (F  ) Hematoxylin and eosin staining of same granuloma at higher magnification, showing numerous cells. (G) Lung sections hybridized with TIMP-1 antisense riboprobe. (H ) High-power magnification showing TIMP-1 transcript in macrophages and mesenchymal cells. (I ) Sections hybridized with TIMP-2 antisense riboprobe. ( J ) Immunohistochemical localization of TIMP-2 in the interior of a silicotic nodule, showing positive macrophages and mesenchymal cells. (A-D, G, I ) Lightly counterstained with eosin; (H and J ) counterstained with hematoxylin.

Regarding TIMP-1 and TIMP-2 expression, label in 60-d silicotic nodules was consistently observed in the peripheral zone of granulomas containing hyaline fibrous tissue in the center (Figures 7G and 7I). Clusters of macrophages were positively stained for both inhibitors as shown for TIMP-1 mRNA (Figure 7H) and TIMP-2 immunoreactive protein (Figure 7J). In addition, positive staining for immunoreactive TIMP-2 is observed in fibroblast-like cells (Figure 7J).

Zymography of Bronchoalveolar Lavage Fluids and Lung Tissue Extracts

For BALF zymograms equal volumes of lavage fluid (8 µl) containing ~ 0.5 µg of protein (controls) and ~ 3 µg of protein (15 and 60 d after silica exposure) were used. Aliquots of controls and experimental BALF were not adjusted by protein concentration; considering that similar volume is recovered after lavage, the higher protein content in experimental lungs is a component of the lung injury. Gelatin zymography showed that both gelatinase A and gelatinase B were present in all BALF samples derived from silica-instilled animals (Figures 8A and 8B). BALF from control rats exhibited a faint band of 72 kD corresponding to progelatinase A (Figure 8A, lanes C). BALF obtained 15 and 60 d after silica instillation showed increased progelatinase A activity, and faint bands representing its activated form (~ 62 kD). Additional bands with apparent molecular sizes of 95 and 86 kD corresponding to progelatinase B and its activated form were present in BALF at 15 and 60 d (Figure 8A, lanes 15 and 60). Higher molecular weight bands of gelatinolytic activity were also evident, mainly in more concentrated samples, and presumably represented lipocalin-associated latent form specific to neutrophils, and/or dimers of the gelatinases (Figure 8B).

View larger version (104K): [in this window] [in a new window]   Figure 8.   Identification of gelatinolytic enzymes in BALF and lung tissue extracts by SDS-PAGE gelatin zymography. (A and B) BALF samples were mixed with an equal volume of Laemmli sample buffer containing 3% SDS. Each lane represents BALF from a different rat. Control rats (lanes C ), and silica-exposed animals after 15 and 60 d; prestained MW standards (left lane). (C ) Aliquots of supernatant from lung extracts. Each lane represents a different rat tissue extract. Control rat (lane C ); silica-exposed animals (lanes 15 and 60); conditioned medium from lung fibroblasts as gelatinase A marker (lane GA); conditioned medium derived from U2-OS cells as marker for gelatinase B (lane GB). Zones of enzymatic activity appear as clear bands over a dark background. Gelatinolytic activity bands were inhibited by EDTA.

In the case of lung tissue extract supernatants, aliquots containing ~ 5 µg of protein (controls and experimental animals) were used. Gelatin zymography showed increased progelatinases A and B 15 and 60 d after silica instillation as compared with controls (Figure 8C). In addition, in the majority of the 15-d samples an additional activated gelatinase A band of ~ 62 kD was observed as exemplified in Figure 8C. EDTA inhibited BALF and lung tissue gelatinolytic activity bands (not shown).

	DISCUSSION

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The pathogenic mechanisms responsible for the progressive collagen accumulation within silicotic granulomas have not been elucidated. Several presumably fibrogenic cytokines have been suggested to play a role in experimental models of silicosis and in the human disease. In this context, it has been shown that tumor necrosis factor  (TNF-) localized in alveolar macrophages (16) is upregulated and, more interestingly, silica- induced fibrosis is almost completely prevented by anti-TNF- antibody (17). On the other hand, transforming growth factor  (TGF-) expression has been shown to be increased in the rat model, associated with a heterogeneous population of cells dispersed throughout the granulomas, mainly macrophages (4). In addition, type 2 pneumocytes have also been implicated (18). In the same model, TGF- was localized in an exact correlation with areas of fibroblast-like cells expressing type I procollagen (4). In human silicosis, TGF- has also been found in macrophages, fibroblasts, and hyperplastic alveolar epithelium (19).

Coincidentally, numerous studies have shown that both cytokines regulate several MMPs and TIMPs. TNF- is able to induce gelatinase B and collagenase, and it has a bifunctional effect on TIMP-1 production, such as stimulation at low concentrations and downregulation at higher concentrations (20). TGF- enhances TIMP-1 production, downregulates collagenase 1 expression, and strongly induces human collagenase 3, a homolog of rat collagenase 3 (24, 25). Conversely, MMP-2 and TIMP-2 do not respond to most known cytokines, and they are, at least in vitro, constitutively expressed by an ample variety of cells (6, 9, 26). However, in some pathological conditions they have been shown to be overexpressed, suggesting that unknown mechanisms upregulate these molecules in vivo (13, 27).

In the present study we examined the temporal pattern expression of collagenase 3, gelatinases A and B, and TIMP-1 and TIMP-2 during the progression of silicosis. Although early and mature nodules representing different stages of progression can be visualized in the same lung after a single intratracheal instillation of silica, we selected two periods of time: 15 d after exposure, representing early, predominantly inflammatory granulomatous lesions, and 60 d after exposure to exemplify mainly late, fibrotic lesions.

Our results showed in early granulomas a marked expression of collagenase 3, and gelatinases A and B. Staining was apparent not only inside granulomas but also in collections of macrophages situated in the alveoli and interstitial tissues around the respiratory bronchioles. Interestingly, MMP expression had decreased by 60 d of evolution, mainly in fibrotic granulomas. Thus some advanced granulomas did not exhibit MMP expression despite a considerable number of cells in them.

In one report a similar finding has been shown for the expression of urokinase-type plasminogen activators (uPAs) during experimental silicosis. uPA mRNA levels displayed a rapid and marked upregulation after silica treatment, followed by a progressive decrease later on (28). Comparable changes have been found in the expression of plasminogen activator inhibitors type 1 and type 2 during the evolution of silicosis in mice lungs (29). These are important observations, because plasminogen activator appears to play a role in limiting fibrosis by disassembling the fibrin and procollagen scaffolds, and also by enhancing MMP activation.

Altered MMP activity, either by higher expression of the enzymes and/or their inhibitors, may have different effects on lung matrix remodeling. Gelatinases A and B upregulated early in the lung parenchyma of silicotic rats may participate in basement membrane disruption, an important pathological event observed in lung lesions evolving into human fibrosis (30). In this context, increased gelatinolytic activity has been found in lung tissues and BALF of patients with idiopathic pulmonary fibrosis, mainly in the early phase (31), as well as in sublethal hyperoxic damage in rats, which may evolve to fibrosis (27).

In experimental silicosis basement membrane disruption has been also documented. Thus, ultrastructural observations have revealed the early presence of breaks in the epithelial basement membranes and discontinuous (or absent) basement membrane in sites where endothelial cells developed sproutlike projections (32). Our findings of enhanced lung gelatinase production, associated with the development of silicosis, strongly suggest that these enzymes may participate in matrix and basement membrane remodeling. However, some differences arise from the comparison of gelatin zymograms from BALF and lung tissue extracts: (1) while in BALF no apparent differences were noticed between 15 and 60 d, in lung extracts stronger activities were observed at 15 d. Presumably, BALF gelatinases are primarily those of free alveolar macrophages and neutrophils that are markedly increased at early and advanced phases of silicosis compared with controls. In contrast, gelatinases from lung extracts reflect the overall activities from silicotic, inflamed, and normal areas of the lung parenchyma; and (2) while in control BALF no gelatinase B was detected, in control lung extracts a band representing progelatinase B activity was usually found. This finding may be explained, at least partially, by the fact that lung extract homogenates include a higher number and variety of cells that are not represented in BALF.

On the other hand, increased interstitial collagenase activity during the early phase, where the inflammatory response develops into granulomas, may not only provoke excessive collagen degradation but may also participate in the release of connective tissue-associated growth factorsi.e., transforming growth factor ₁thus enhancing extracellular matrix accumulation (33).

In contrast, the decreased expression of MMPs noticed in the advanced phases of silicosis may contribute to collagen accumulation, and the development of progressive fibrosis. Indeed, these results confirm older findings suggesting that early fibrogenesis is characterized by an increase in collagen synthesis as well as collagenase activity, whereas in more advanced stages a progressive decrease in collagenolytic activity is usually observed (2, 34). The findings of the present study suggest that this process is at least partially associated with changes in collagenase expression, which is markedly increased in early stages whereas it shows a concomitant decrease in late stages.

Alveolar macrophages, which have been appointed as a pivotal cell type in fibrogenesis, were a major cell source of the three MMPs investigated. It has been suggested that the expression of matrix-degrading proteases regulates a variety of macrophage functions including extravasation, migration and tissue remodeling (35).

Regarding TIMPs, there was also a substantial increase at 15 d with a moderate reduction at 60 d when compared with MMPs. The possible role(s) of these inhibitors during the progression to fibrotic nodules is at present unclear, but one obvious effect is related to their ability to inhibit MMPs. However, the functional relationships and interactions between TIMPs and MMPs are intricate, and despite the fact that TIMPs share a common inhibitory activity for all members of the MMP family they also seem to have specific functions. For example, TIMP-1 and TIMP-2 form preferential complexes with progelatinase B and progelatinase A, respectively, through interactions that are different from those between TIMPs and the active form of these enzymes (38, 39). These complexes, such as progelatinase B/TIMP-1 and progelatinase A/TIMP-2, control the rate at which physiological factors activate MMPs.

In addition, TIMP-1 and TIMP-2 may participate in other functions, because it has been shown that they are able to promote the proliferation of a variety of malignant and normal cells, including fibroblasts (40, 41). Therefore, in the inflamed lung microenvironment, the upregulation of TIMP-1 and TIMP-2 may affect matrix remodeling not only by controlling enzyme activity but also by stimulating mesenchymal cell proliferation.

In conclusion, the in vivo observations discussed here demonstrate that several MMPs and TIMPs are upregulated during the development of silicosis, and suggest that changes in the expression of interstitial collagenase and gelatinases A and B are implicated in extracellular matrix remodeling and basement membrane disruption. Continuous upregulation of TIMP-1 and TIMP-2 may play a dual role: on the one hand to enhance MMP inhibition, mainly in advanced stages, and on the other hand to stimulate fibroblast proliferation. Further studies regarding the physiological role in vivo of extracellular matrix-remodeling molecules are necessary to understand the molecular basis of continuous matrix accumulation.